1. Field of the Invention
The present invention relates generally to viral variants exhibiting reduced sensitivity to particular agents and/or reduced interactivity with immunological reagents. More particularly, the present invention is directed to hepatitis B virus (HBV) variants exhibiting complete or partial resistance to nucleoside or nucleotide analogs or other antagonists of HBV DNA polymerase activity and/or reduced interactivity with antibodies to viral surface components including reduced sensitivity to these antibodies. The present invention further contemplates assays for detecting such viral variants, which assays are useful in monitoring anti-viral therapeutic regimens and in developing new or modified vaccines directed against viral agents and in particular the resistant HBV variants of the present invention. The present invention also contemplates the use of the viral variants to screen for agents capable of inhibiting infection, replication and/or release of the virus.
2. Description of the Prior Art
Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.
The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in any country.
Specific mutations in an amino acid sequence are represented herein as ‘Xaa1nXaa2’ where Xaa1 is the original amino acid residue before mutation, n is the residue number and Xaa2 is the mutant amino acid. The abbreviation ‘Xaa’ may be the three letter or single letter (i.e. ‘X’) code. The amino acid residues for Hepatitis B virus DNA polymerase are numbered with the residue methionine in the motif Tyr Met Asp Asp (YMDD) being residue number 204 (Stuyver et al., Hepatology 33: 751-757, 2001). The amino acid residues for hepatitis B virus surface antigen are number according to Norder et al. (J. Gen. Virol. 74: 341-1348, 1993).
The term nucleoside analogs has been used in reference to both nucleotide and nucleoside analogs.
Hepatitis B virus (HBV) can cause debilitating disease conditions and can lead to acute liver failure. HBV is a DNA virus which replicates via an RNA intermediate and utilizes reverse transcription in its replication strategy (Summers and Mason, Cell 29: 403-415, 1982). The HBV genome is of a complex nature having a partially double-stranded DNA structure with overlapping open reading frames encoding surface, core, polymerase and X genes. The complex nature of the HBV genome is represented in FIG. 1. The polymerase consists of four functional regions, the terminal protein (TP), spacer, reverse transcriptase (rt) and ribonuclease (RNAse).
The polymerase gene of HBV overlaps the envelope gene, mutations in the catalytic domain of the polymerase can affect the amino acid sequence of the envelope protein and vice versa. In particular, the genetic sequence for the neutralization domain of HBV known as the ‘a’ determinant, which is found within the HBsAg and located between amino acids 99 and 169, actually overlaps the major catalytic regions of the viral polymerase protein and in particular domains A and B.
The presence of an HBV DNA polymerase has led to the proposition that nucleoside and nucleotide analogs could act as effective anti-viral agents. Examples of nucleoside analogs currently being tested are penciclovir and its oral form (FAM or FCV) [Vere Hodge, Antiviral Chem Chemother 4: 67-84, 1993; Boyd et al., Antiviral Chem Chemother. 32: 358-363, 1987; Kruger et al., Hepatology 22: 219A, 1994; Main et al., J. Viral Hepatitis 3: 211-215, 1996] Lamivudine [(−)-β-2′-deoxy-3′-thiacytidine; (3TC or LMV) [Severini et al., Antimicrobial Agents Chemother 39: 1430-1435, 1995; Dienstag et al., New England J Med 333: 1657-1661, 1995]. New nucleoside analogs which have already progressed to clinical trials include the pyriamidines Emtricitabine, ((−)-β-L-2′-3′-dideoxy-5-fluoro-3′-thiacydidine; FTC), the 5-fluoro derivative of 3TC, and Clevudine (1-(2-fluoro-5-methyl-β-L-arabino-furanosyl) uracil; L-FMAU), a thymidine analog. Like 3TC, these are pyrimidine derivatives with an unnatural “L”-configuration. Several purine derivatives have also progressed to clinical trials; they include Entecavir (BMS-200,475; ETV), a carbocyclic deoxyguanosine analog, diaminopurine dioxolane (DAPD), an oral pro-drug for dioxolane guanine ((−)-β-D-2-aminopurine dioxolane; DXG) and Adefovir dipivoxil, an oral prodrug for the acyclic deoxyadenosine monophosphate nucleoside analog Adefovir (9-[phosphonyl-methoxyethyl]-adenine; PMEA). Tenofovir disoproxil fumarate (TDF) has activity against HBV in vitro [Lada O et al., Antivir Ther.; 9:353-63, 2004]. TDF has been approved for the treatment of HIV and has been used in co-infected HIV-HBV patients for the treatment of both HBV and HIV.
Whilst these agents are highly effective in inhibiting HBV DNA synthesis, there is the potential for resistant mutants of HBV to emerge during long term antiviral chemotherapy. In patients on prolonged LMV therapy key resistance mutations are selected in the rt domain within the polymerase at rtM204I/V+/−rtL180M. The nomenclature used for the polymerase mutations is in accordance with that proposed by Stuyver et al., 2001, supra. Only LMV and ADV have been approved for use against chronic HBV infection. LMV and ADV are both potent inhibitors of HBV replication and reduce HBV DNA viral load. Unfortunately, both LMV-resistant and ADV-resistant strains of HBV are selected during therapy.
HIV and HBV are both blood borne viruses and have similar potential routes of transmission. Approximately 10% of HIV infected individuals are co-infected with HBV. The improved prognosis of HIV-infection that has occurred since the introduction of highly active antiretroviral therapy (HAART) has resulted in renewed emphasis being placed on co-morbidities associated with HIV-infection, and chronic viral hepatitis in particular. Hepatitis B virus (HBV) infection is an important infection in HIV-1 infected individuals because of the influence of HIV-1 co-infection on the natural history of HBV infection. Antiviral therapies with activity against both viruses have enabled targeted therapy in co-infected individuals. However, antiviral resistance is an important issue for both HIV and HBV.
TDF is also a potent inhibitor of HBV replication. TDF has activity against both hepadnaviruses and HIV. TDF was formerly referred to as bis-POC PMPA the oral derivative of 9-[(R)-2-(phosphonomethoxy)propyl]adenine (PMPA) [Shaw J P et al., Pharm Res. 14(12):1824-9, 1997]. Preclinical studies demonstrate that TDF is a highly potent inhibitor of HBV in vitro and is effective against the LMV resistant viruses [Ying C et al., J Viral Hepat. 7(2):161-5, 2000]. TDF has been used successfully to treat patients with the LMV resistant HBV mutations. No specific TDF resistant mutations had been described for HBV.
Nucleoside or nucleotide analog therapy may be administered as monotherapy or combination therapy where two or more nucleoside or nucleotide analogs may be administered. There is a need to monitor for development of nucleoside-/nucleotide-resistant variants of HBV as well as variants resistant to other HBV DNA polymerase antagonists and variants resistant to antibodies to HBV surface components. The rapid identification can lead to altered therapeutic protocols being pursued. In addition, resistant HBV variants are useful as targets for screening of new antiviral agents.